Pancreatic ductal adenocarcinoma is the fourth leading cause of cancer-related death in the U.S. (1). It is notably the most aggressive and debilitating malignant disease with a median survival of less than 6 months. Only 1% to 4% of patients have an overall survival of more than 5 years (1). Inadequate early diagnosis, resistance to current therapies, and ineffective treatment account for these low survival statistics. Alternative treatment approaches are desparately needed for this disease; the compelling need for superior treatment options has propelled the development of new, better-targeted therapies. We have developed an allogeneic, granulocyte-macrophage colony-stimulating factor (GM-CSF)-secreting pancreatic cancer vaccine, which has recently completed phase II clinical trial (2). This promising vaccine is used in combination with chemoradiation. The observation of favorable clinical and immunological responses in patients has testified to the success of the vaccine (2-4). It was shown that the induction of mesothelin-specific T cell responses only in patients with a DFS>3 years, which suggests the vaccine induces immunologically relevant T cell responses (2). Functional genomic approaches were utilized to identify antigens recognized by T cells (5). However, finding T cell antigens is limited by the need for large amounts of patient lymphocytes and the lack of reagents for each patient-specific HLA (6).
In contrast to T cells, antibodies hold potential as a high throughput way of identifying antigens. Antibodies can also mount an effective response against cancer cells through opsonizing, antigen presentation to T-cells, and mediating cell toxicity via natural killer cells or the complement system (7). Thus, the application of seroproteomic approaches has recently gained ground in the identification of new cancer biomarkers. These cancer biomarkers are beneficial for both early detection and the determination of new targets for the development of biologically relevant therapies (7-12). The most well-known proteomic approaches utilize sera from untreated cancer patients or individuals with known genetic susceptibilities for cancer, to screen for cancer-associated proteins that elicit an antibody response. These approaches identify oncoproteins that elicit an antibody response due to differences in expression levels or post-translational modifications (11). GM-CSF secreting cancer vaccines can also instigate a broad range of antibody responses, as seen in early clinical studies (13). Through the study of the immunological responses in vaccinated patients, we can discover the mechanisms behind favorable vaccine-induced clinical responses. Identifying cancer associated proteins will enhance our efforts of identifying biologically relevant proteins. These proteins have high potential as future targets for effective pancreatic cancer treatment. This translational approach will advance the development of new drugs, vaccines and antibody-based therapies that will halt the progression and metastasis of the disease. This approach can also help characterize new proteins that will serve as surrogate biomarkers, prediction tools of the vaccine's success, and biomarkers for early diagnosis of pancreatic cancer.
Common proteomic approaches to identify immunogenic proteins are: Serological Screening of cDNA Expression Library (SEREX), 2-dimensional electrophoresis (2-DE) followed by mass-spectrometry analysis and protein arrays (7). Proteins found using SEREX and 2-DE approaches are now shown to also elicit T cell responses (6, 13, 14). This provides evidence that antibodies can aid in the identification of T cell antigens, which further testifies to the advantages in studying antibodies. SEREX, however, utilizes proteins expressed in Escherichia coli, which does not account for human post-translational modifications (12). Contrastingly, the approach utilizing 2-DE analysis can use human proteins as the proteome. However, this process has an inherent bias towards identifying proteins that are abundantly expressed (11). 2D-PAGE has a lower threshold out of the throughput methods and does not effectively identify proteins that are very acidic, very basic, small in size (<15 kDa), or hydrophobic (15). Therefore, this process is inadequate for detecting membrane-associated proteins, the most relevant category of proteins as potential biomarkers. Membrane proteins constitute about 30% of all cellular proteins and are functionally key regulators (16). In addition, in 2D-PAGE, each band cut holds several similar molecular weight proteins. This process is inefficient in separating single proteins, which obscures which protein instigates the antibody response. Furthermore, low abundant antigens are generally overshadowed by high abundant proteins with the same molecular weight in this process. Both SEREX and SERPA identify linear epitopes, are relatively low throughput and semi-quantitative (11). Protein arrays come in many forms. Some protein arrays use tumor cell lysate fractions, which identify proteins in their native conformation (11). However, these arrays do not identify which specific protein in the fraction instigates the immune response and there also issues with fractionation. The protein arrays with printed recombinant proteins do not contain human post-translational modifications because the proteins are expressed in E. coli or yeast (12). In addition, if a known protein panel is printed, tumor antigen discovery can be prevented because the proteome is biased. The protein arrays utilizing printed monoclonal antibodies are potentially limited by reagent availability thereby preventing an unbiased proteome being used because a high affinity and highly specific monoclonal antibody is needed for each protein to be probed.
There is a continuing need in the art to provide better methods of early diagnosis, monitoring, prognosing, and treating pancreatic cancer.